Purification and characterization of a cellulase from the ruminal fungus Orpinomyces joyonii cloned in Escherichia coli

A cellulase from the ruminal fungus Orpinomyces joyonii cloned in Escherichia coli was purified 88-fold by chromatography on High Q and hydroxyapatite. N-terminal amino acid sequence analyses confirmed that the cellulase represented the product of the cellulase gene Cel B2. The purified enzyme possessed high activity toward barley beta-glucan, lichenan, carboxymethyl cellulose (CMC), xylan, but not toward laminarin and pachyman. In addition, the cloned enzyme was able to hydrolyze p-nitrophenyl (PNP)-cellobioside, PNP-cellotrioside, PNP-cellotetraoside, PNP-cellopentaoside, but not PNP-glucopyranoside. The specific activity of the cloned enzyme on barley beta-glucan was 297 units/mg protein. The purified enzyme appeared as a single band in SDS-polyacrylamide gel electrophoresis and the molecular mass of this enzyme (58000) was consistent with the value (56463) calculated from the DNA sequence. The optimal pH of the enzyme was 5.5, and the enzyme was stable between pH 5.0 and pH 7.5. The enzyme had a temperature optimum at 40 degrees C. The K(m) values estimated for barley beta-glucan and CMC were 0.32 and 0.50 mg/ml, respectively.     

A variant of Orpinomyces joyonii 1,3-1,4-beta-glucanase with increased thermal stability obtained by random mutagenesis and screening

A thermostable variant of an Orpinomyces joyonii beta-glucanase was identified by screening a mutant library constructed using error-prone PCR products. The mutant, designated 2011D, had one amino acid substitution (Val replaced Asp-70). 2011D showed similar catalytic efficiency to its wild-type enzyme, LicA. The temperature at which 50% inactivation occurred after heat treatment for 10 min was increased by 14 degrees C for 2011D, in comparison to those of wild-type enzyme.     

Cloning and overexpression of a Paenibacillus beta-glucanase in Pichia pastoris: purification and characterization of the recombinant enzyme

Isolation, expression, and characterization of a novel endo-beta-1,3(4)-D-glucanase with high specific activity and homology to Bacillus lichenases is described. One clone was screened from a genomic library of Paenibacillus sp. F-40, using lichenan-containing plates. The nucleotide sequence of the clone contains an ORF consisting of 717 nucleotides, encoding a beta-glucanase protein of 238 amino acids and 26 residues of a putative signal peptide at its N-terminus. The amino acid sequence showed the highest similarity of 87% to other beta-1,3-1,4-glucanases of Bacillus. The gene fragment Bg1 containing the mature glucanase protein was expressed in Pichia pastoris at high expression level in a 3-1 high-cell-density fermenter. The purified recombinant enzyme Bgl showed activity against barley beta-glucan, lichenan, and laminarin. The gene encodes an endo-beta-1,3(4)-D-glucanase (E. C. 3.2.1.6). When lichenan was used as substrate, the optimal pH was 6.5, and the optimal temperature was 60 degrees C. The K(m), V(max), and k(cat) values for lichenan are 2.96 mg/ml, 6,951 micromol/min x mg, and 3,131 s(-1), respectively. For barley beta-glucan the values are 3.73 mg/ml, 8,939 micromol/min x mg, and 4,026 s(-1), respectively. The recombinant Bg1 had resistance to pepsin and trypsin. Other features of recombinant Bg1 including temperature and pH stability, and sensitivity to some metal ions and chemical reagents were also characterized.     

Cloning and expression of a Bacillus subtilis Endo-1,3-1,4-beta-D-glucanase gene in Escherichia coli K12

EcoRI fragments of DNA from Bacillus subtilis NCIB 8565, a high producer of an endo-1,3-1,4-beta-D-glucanase, were 'shot-gun' cloned in the plasmid vector pBR325. A 3.5 kb insert, carrying single restriction sites for AvaI, BglII, ClaI, PvuI and PvuII, was shown to direct the synthesis of beta-glucanase in Escherichia coli K12. Enzyme activity was demonstrated in extracellular fractions of E. coli harbouring the beta-glucanase gene; however, the largest proportion (greater than 50%) of total enzyme activity was periplasmic in location. beta-Glucanase activity and cellular location were independent of the orientation of the 3.5 kb fragment in pBR325.     

产β—葡聚糖酶菌种T199的选育及发酵条件

大麦为啤酒酿造原料 ,含有由葡萄糖残基通过β 1 ,3 和 1 ,4 糖甙键连接而成的β 葡聚糖。在麦芽汁制备过程中 ,热不稳定的大麦葡聚糖酶不能充分降解β 葡聚糖 ,残留的 β 葡聚糖不仅影响麦芽汁分离和啤酒过滤 ,而且将成为成品啤酒出现混浊和沉淀的因素之一。微生物 β 葡聚糖酶能改善啤酒加工工艺和提高产品质量[1,2 ] 。谷类饲料含有不同于纤维素的 β 葡聚糖[2 ] ,作为抗营养因子 ,β 葡聚糖使饲料具有粘性 ,不能很好的消化利用。β 葡聚糖酶作为饲料添加剂加入到饲料中 ,可以将 β 葡聚糖降解 ,从而提高饲料利用率 ,改善营养吸收。相关…     

Synthesis and secretion of a Bacillus circulans WL-12 1,3-1,4-beta-D-glucanase in Escherichia coli.

The synthesis and secretion of a 1,3-1,4-beta-D-glucanase were studied in different strains of Escherichia coli transformed with plasmids carrying the Bacillus circulans WL-12 1,3-1,4-beta-D-glucanase structural gene. This gene (named BGC) is contained within a 1.9-kilobase BamHI-HindIII fragment and directs the synthesis in E. coli of an enzyme that specifically degrades lichenan. Only one active form of the enzyme was found when the gene was expressed in different E. coli strains. The electrophoretic pattern of this protein showed a molecular weight that was approximately the same as that of the mature beta-glucanase secreted from B. circulans WL-12, suggesting that the processing of this protein may be similar in both species. As deduced from maxicell experiments, the Bacillus parental promoter directs the synthesis in E. coli. Pulse-chase experiments showed that the protein may be cotranslationally processed.     

Purification and properties of a 1,3-1,4-beta-D-glucanase (lichenase, 1,3-1,4-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.73) from Bacteroides succinogenes cloned in Escherichia coli.

A 1,3-1,4-beta-D-glucanase (lichenase, 1,3-1,4-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.73) from Bacteroides succinogenes cloned in Escherichia coli was purified 600-fold by chromatography on Q-Sepharose and hydroxyapatite. The cloned enzyme hydrolysed lichenin and oat beta-D-glucan but not starch, CM(carboxymethyl)-cellulose, CM-pachyman, laminarin or xylan. The enzyme had a broad pH optimum with maximum activity at approx. pH 6.0 and a temperature optimum of 50 degrees C. The pH of elution from a chromatofocusing column for the cloned enzyme was 4.7 (purified) and 4.9 (crude) compared with 4.8 for the mixed-linkage beta-D-glucanase activity in B. succinogenes. The Mr of the cloned enzyme was estimated to be 37,200 by gel filtration and 35,200 by electrophoresis. The Km values estimated for lichenin and oat beta-D-glucan were 0.35 and 0.71 mg/ml respectively. The major hydrolytic products with lichenin as substrate were a trisaccharide (82%) and a pentasaccharide (9.5%). Hydrolysis of oat beta-D-glucan yielded a trisaccharide (63.5%) and a tetrasaccharide (29.6%) as the major products. The chromatographic patterns of the products from the cloned enzyme appear to be similar to those reported for the mixed-linkage beta-D-glucanase isolated from Bacillus subtilis. The data presented illustrate the similarity in properties of the cloned mixed-linkage enzyme and the 1,3-1,4-beta-D-glucanase from B. subtilis and the similarity with the 1,4-beta-glucanase in B. succinogenes.     

Essential catalytic role of Glu134 in endo-beta-1,3-1,4-D-glucan 4-glucanohydrolase from B. licheniformis as determined by site-directed mutagenesis.

Site-directed mutagenesis experiments designed to identify the active site of Bacillus licheniformis endo-beta-1,3-1,4-D-glucan 4-glucanohydrolase (beta-glucanase) have been performed. Putative catalytic residues were chosen on the basis of sequence similarity analysis to viral and eukaryotic lysozymes. Four mutant enzymes were expressed and purified from recombinant E. coli and their kinetics analysed with barley beta-glucan. Replacement of Glu134 by Gln produced a mutant (E134Q) that retains less than 0.3% of the wild-type activity. The other mutants, D133N, E160Q and D179N, are active but show different kinetic parameters relative to wild-type indicative of their participation in substrate binding and transition-state complex stabilization. Glu134 is essential for activity; it is comprised in a region of high sequence similarity to the active site of T4 lysozyme and matches the position of the general acid catalyst. These results strongly support a lysozyme-like mechanism for this family of Bacillus beta-glucan hydrolases with Glu134 being the essential acid catalyst.     

A novel beta-glucanase gene from Bacillus halodurans C-125

A novel endo-beta-1,3(4)-D-glucanase gene was found in the complete genome sequence of Bacillus halodurans C-125. The gene was previously annotated as an "unknown" protein and assigned an incorrect open reading frame (ORF). However, determining the biochemical characteristics has elucidated the function and correct ORF of the gene. The gene encodes 231 amino acids, and its calculated molecular mass was estimated to be 26743.16 Da. The amino acid sequence alignment showed that the highest sequence identity was only 28% with that of the beta-1,3-1,4-glucanase from Bacillus subtilis. Moreover, the nucleotide sequence did not match any other known Bacillus beta-glucanase gene. The member of the gene cluster that includes this novel gene was apparently different from that of the gene cluster including the putative beta-glucanase genes (bh3231 and bh3232) from B. halodurans C-125. Therefore, the novel gene is not a copy of either of these genes, and in B. halodurans cells, the putative role of the encoded protein may differ from that of bh3231 and bh3232. To examine the activity of the gene product, the gene was cloned as a His-tagged protein and expressed in Escherichia coli. The purified enzyme showed activity against lichenan, barley beta-glucan, laminarin, and carboxymethyl curdlan. Thin-layer chromatography showed that the enzyme hydrolyzes substrates in an endo-type manner. When beta-glucan was used as a substrate, the pH optimum was between 6 and 8, and the temperature optimum was 60 degrees C. After 2 h incubation at 50 and 60 degrees C, the residual activity remained 100% and 50%, respectively. The enzymatic activity was abolished after 30 min incubation at 70 degrees C. Based on the results, the gene encodes an endo-type beta-1,3(4)-D-glucanase (E.C. 3.2.1.6).     

Characterization of an acetyl xylan esterase from the anaerobic fungus Orpinomyces sp. strain PC-2.

A 1,067-bp cDNA, designated axeA, coding for an acetyl xylan esterase (AxeA) was cloned from the anaerobic rumen fungus Orpinomyces sp. strain PC-2. The gene had an open reading frame of 939 bp encoding a polypeptide of 313 amino acid residues with a calculated mass of 34,845 Da. An active esterase using the original start codon of the cDNA was synthesized in Escherichia coli. Two active forms of the esterase were purified from recombinant E. coli cultures. The size difference of 8 amino acids was a result of cleavages at two different sites within the signal peptide. The enzyme released acetate from several acetylated substrates, including acetylated xylan. The activity toward acetylated xylan was tripled in the presence of recombinant xylanase A from the same fungus. Using p-nitrophenyl acetate as a substrate, the enzyme had a K(m) of 0.9 mM and a V(max) of 785 micromol min(-1) mg(-1). It had temperature and pH optima of 30 degrees C and 9.0, respectively. AxeA had 56% amino acid identity with BnaA, an acetyl xylan esterase of Neocallimastix patriciarum, but the Orpinomyces AxeA was devoid of a noncatalytic repeated peptide domain (NCRPD) found at the carboxy terminus of the Neocallimastix BnaA. The NCRPD found in many glycosyl hydrolases and esterases of anaerobic fungi has been postulated to function as a docking domain for cellulase-hemicellulase complexes, similar to the dockerin of the cellulosome of Clostridium thermocellum. The difference in domain structures indicated that the two highly similar esterases of Orpinomyces and Neocallimastix may be differently located, the former being a free enzyme and the latter being a component of a cellulase-hemicellulase complex. Sequence data indicate that AxeA and BnaA might represent a new family of hydrolases.     

Activity staining of cellulases in polyacrylamide gels containing mixed linkage beta-glucans

Endoglucanase and cellobiohydrolase components of thermophilic cellulases can be detected in situ after gel electrophoresis in the presence of sodium dodecyl sulfate by incorporating a mixed linkage beta-glucan (barley beta-glucan, lichenan) in the separation gel. Zymograms are prepared after a renaturation treatment and incubation by staining the gel with Congo red. This method is suitable for the detection of beta-glucanases with different substrate specificities cleaving beta-1,4-, beta-1,4-1,3-, or beta-1,3-glucans. Cellobiohydrolase activities can be detected by adding 4-methylumbelliferyl-beta-D-cellobioside to the incubation buffer. The gels are subsequently stained with Coomassie blue to establish identical molecular weights of beta-glucanase and protein bands. Applications of this technique for the comparison of cellulases and for the identification of cellulase components expressed from recombinant clones are presented.     

Structure of the cel-3 gene from Fibrobacter succinogenes S85 and characteristics of the encoded gene product, endoglucanase 3.

The cel-3 gene cloned from Fibrobacter succinogenes into Escherichia coli coded for the enzyme EG3, which exhibited both endoglucanase and cellobiosidase activities. The gene had an open reading frame of 1,974 base pairs, coding for a protein of 73.4 kilodaltons (kDa). However, the enzyme purified from the osmotic shock fluid of E. coli was 43 kDa. The amino terminus of the 43-kDa protein matched amino acid residue 266 of the protein coded for by the open reading frame, indicating proteolysis in E. coli. In addition to the 43-kDa protein, Western immunoblotting revealed a 94-kDa membranous form of the enzyme in E. coli and a single protein of 118 kDa in F. succinogenes. Thus, the purified protein appears to be a proteolytic degradation product of a native protein which was 94 kDa in E. coli and 118 kDa in F. succinogenes. The discrepancy between the molecular weight expected on the basis of the DNA sequence and the in vivo form may be due to anomalous migration during electrophoresis, to glycosylation of the native enzyme, or to fatty acyl substitution at the N terminus. One of two putative signal peptide cleavage sites bore a strong resemblance to known lipoprotein leader sequences. The purified 43-kDa peptide exhibited a high Km (53 mg/ml) for carboxymethyl cellulose but a low Km (3 to 4 mg/ml) for lichenan and barley beta-glucan. The enzyme hydrolyzed amorphous cellulose, and cellobiose and cellotriose were the major products of hydrolysis. Cellotriose, but not cellobiose, was cleaved by the enzyme. EG3 exhibited significant amino acid sequence homology with endoglucanase CelC from Clostridium thermocellum, and as with both CelA and CelC of C. thermocellum, it had a putative active site which could be aligned with the active site of hen egg white lysozyme at the highly conserved amino acid residues Asn-44 and Asp-52.     

DNA sequence of a Fibrobacter succinogenes mixed-linkage beta-glucanase (1,3-1,4-beta-D-glucan 4-glucanohydrolase) gene.

The DNA sequence of a mixed-linkage beta-glucanase (1,3-1,4-beta-D-glucan 4-glucanohydrolase [EC 3.2.1.73]) gene from Fibrobacter succinogenes cloned in Escherichia coli was determined. The general features of this gene are very similar to the consensus features for other gram-negative bacterial genes. The gene product was processed for export in E. coli. There is a high level of sequence homology between the structure of this glucanase and the structure of a mixed-linkage beta-glucanase from Bacillus subtilis. The nonhomologous region of the amino acid sequence includes a serine-rich region containing five repeats of the sequence Pro-Xxx-Ser-Ser-Ser-Ser-(Ala or Val) which may be functionally related to the serine-rich region observed in Pseudomonas fluorescens cellulase and the serine- and/or threonine-rich regions observed in Cellulomonas fimi endoglucanase and exoglucanase, in Clostridium thermocellum endoglucanases A and B, and in Trichoderma reesei cellobiohydrolase I, cellobiohydrolase II, and endoglucanase I.     

Two noncellulosomal cellulases of Clostridium thermocellum, Cel9I and Cel48Y, hydrolyse crystalline cellulose synergistically

The genome of Clostridium thermocellum contains a number of genes for polysaccharide degradation-associated proteins that are not cellulosome bound. The list includes beta-glucanases, glycosidases, chitinases, amylases and a xylanase. One of these 'soluble'-enzyme genes codes for a second glycosyl hydrolase (GH)48 cellulase, Cel48Y, which was expressed in Escherichia coli and biochemically characterized. It is a cellobiohydrolyse with activity on native cellulose such as microcrystalline and bacterial cellulose, and low activity on carboxymethylcellulose. It is about 100 times as active on amorphic cellulose and mixed-linkage barley beta-glucan compared with cellulase Cel9I. The enzyme Cel48Y shows a distinct synergism of 2.1 times with the noncellulosomal processive endoglucanase Cel9I on highly crystalline bacterial cellulose at a 17-fold excess of Cel48Y over Cel9I. These data show that C. thermocellum has, besides the cellulosome, the genes for a second cellulase system for the hydrolysis of crystalline cellulose that is not particle bound.     

Isolation and analysis of two cellulase cDNAs from Orpinomyces joyonii

Two cellulase cDNAs, celB29 and celB2, were isolated from a cDNA library derived from mRNA extracted from the anaerobic fungus, Orpinomyces joyonii strain SG4. The nucleotide sequences of celB2 and celB29 and the primary structures of the proteins encoded by these cDNAs were determined. The larger celB29 cDNA was 1966bp long and encoded a 477 amino acid polypeptide with a molecular weight of 54kDa. Analysis of the 1451bp celB2 cDNA revealed an 1164bp open reading frame coding for a 44kDa protein consisting of 388 amino acids. Both deduced proteins had a high sequence similarity in central regions containing putative catalytic domains. Primary structure analysis revealed that CelB29 contained a Thr/Pro-rich sequence that separated the N-terminal catalytic domain from a C-terminal reiterated region of unknown function. Homology analysis showed that both enzymes belong to glycosyl hydrolase family 5 and were most closely related to endoglucanases from the anaerobic fungi Neocallimastic patriciarum, Neocallimastix frontalis and Orpinomyces sp. The classification of CelB29 and CelB2 as endoglucanases was supported by enzyme assays. The cloned enzymes had high activities towards barley beta-glucan, lichenan and carboxymethylcellulose (CMC), but not Avicel, laminarin, pachyman, xylan and pullulan. In addition, CelB29 and CelB2 showed activity against p-nitrophenyl-beta-D-cellobioside (pNP-G(2)) to p-nitrophenyl-beta-D-cellopentaoside (pNP-G(5)) but not p-nitrophenyl-beta-D-glucopyranoside (pNP-G(1)) with preferential activity against p-nitrophenyl-beta-D-cellotrioside (pNP-G(3)). Based on these results, we proposed that CelB29 and CelB2 are endoglucanases with broad substrate specificities for short- and long-chain beta-1,4-glucans.     

Construction, analysis, and beta-glucanase screening of a bacterial artificial chromosome library from the large-bowel microbiota of mice

A metagenomic (community genomic) library consisting of 5,760 bacterial artificial chromosome clones was prepared in Escherichia coli DH10B from DNA extracted from the large-bowel microbiota of BALB/c mice. DNA inserts detected in 61 randomly chosen clones averaged 55 kbp (range, 8 to 150 kbp) in size. A functional screen of the library for beta-glucanase activity was conducted using lichenin agar plates and Congo red solution. Three clones with beta-glucanase activity were detected. The inserts of these three clones were sequenced and annotated. Open reading frames (ORF) that encoded putative proteins with identity to glucanolytic enzymes (lichenases and laminarinases) were detected by reference to databases. Other putative genes were detected, some of which might have a role in environmental sensing, nutrient acquisition, or coaggregation. The insert DNA from two clones probably originated from uncultivated bacteria because the ORF had low sequence identity with database entries, but the genes associated with the remaining clone resembled sequences reported in Bacteroides species.     

Properties of a thermoactive beta-1,3-1,4-glucanase (lichenase) from Clostridium thermocellum expressed in Escherichia coli

A Clostridium thermocellum gene (licB) encoding a thermoactive 1,3-1,4-beta-glucanase (lichenase) with a molecular weight of about 35,000 was localized on a 1.5-kb DNA fragment by cloning and expression in E. coli. The enzyme acts on beta-glucans with alternating beta-1,3- and beta-1,4-linkages such as barley beta-glucan and lichenan, but not on beta-glucans containing only 1,3- or 1,4-glucosidic bonds. It is active over a broad pH range (pH 5-12) and has a temperature optimum around 80 degrees C. The C. thermocellum lichenase is unusually resistant against inactivation by heat, ethanol or ionic detergents. These properties make the enzyme highly suitable for industrial application in the mashing process of beer brewing.     

A mannanase, ManA, of the polycentric anaerobic fungus Orpinomyces sp. strain PC-2 has carbohydrate binding and docking modules

The anaerobic fungus Orpinomyces sp. strain PC-2 produces a broad spectrum of glycoside hydrolases, most of which are components of a high molecular mass cellulosomal complex. Here we report about a cDNA (manA) having 1924 bp isolated from the fungus and found to encode a polypeptide of 579 amino acid residues. Analysis of the deduced sequence revealed that it had a mannanase catalytic module, a family 1 carbohydrate-binding module, and a noncatalytic docking module. The catalytic module was homologous to aerobic fungal mannanases belonging to family 5 glycoside hydrolases, but unrelated to the previously isolated mannanases (family 26) of the anaerobic fungus Piromyces. No mannanase activity could be detected in Escherichia coli harboring a manA-containing plasmid. The manA was expressed in Saccharomyces cerevisiae and ManA was secreted into the culture medium in multiple forms. The purified extracellular heterologous mannanase hydrolyzed several types of mannan but lacked activity against cellulose, chitin, or beta-glucan. The enzyme had high specific activity toward locust bean mannan and an extremely broad pH profile. It was stable for several hours at 50 degrees C, but was rapidly inactivated at 60 degrees C. The carbohydrate-binding module of the Man A produced separately in E. coli bound preferably to insoluble lignocellulosic substrates, suggesting that it might play an important role in the complex enzyme system of the fungus for lignocellulose degradation.     

Comparative modeling of the three-dimensional structures of family 3 glycoside hydrolases

There are approximately 100 known members of the family 3 group of glycoside hydrolases, most of which are classified as beta-glucosidases and originate from microorganisms. The only family 3 glycoside hydrolase for which a three-dimensional structure is available is a beta-glucan exohydrolase from barley. The structural coordinates of the barley enzyme is used here to model representatives from distinct phylogenetic clusters within the family. The majority of family 3 hydrolases have an NH(2)-terminal (alpha/beta)(8) barrel connected by a short linker to a second domain, which adopts an (alpha/beta)(6) sandwich fold. In two bacterial beta-glucosidases, the order of the domains is reversed. The catalytic nucleophile, equivalent to D285 of the barley beta-glucan exohydrolase, is absolutely conserved across the family. It is located on domain 1, in a shallow site pocket near the interface of the domains. The likely catalytic acid in the barley enzyme, E491, is on domain 2. Although similarly positioned acidic residues are present in closely related members of the family, the equivalent amino acid in more distantly related members is either too far from the active site or absent. In the latter cases, the role of catalytic acid is probably assumed by other acidic amino acids from domain 1.